Three highly expressed proteins involved in light signaling in retinal rod photoreceptors, transducin, recoverin and arrestin, localize to different photoreceptor compartments depending on the conditions of ambient illumination. This transport is important for adjusting photoreceptor light sensitivity. Moreover, it plays a role in protecting rod photoreceptors from damage caused by continual stimulation from light levels we experience every day, and from aberrant photoreceptor activity that may underlie many congenital retinal degenerative diseases. Yet the mechanisms by which these proteins are localized to photoreceptor compartments, by which they are transported among compartments and by which changes in ambient light levels initiate this re-localization are not known. We have developed new methods using multiphoton microscopy to directly examine local, compartment-specific behavior of the proteins and local changes in signaling molecules within living, functioning photoreceptors. Using these new methods we will examine the: Aim 1: Mechanisms underlying protein localization to rod photoreceptor compartments. Aim 2: Mode of signal-dependant protein transport between rod compartments. Aim 3: Signals that initiate protein transport. Experiments in aims 1 and 2 will quantitatively examine the local and long distance mobilities of the proteins fused with a variant of the green fluorescent protein, photoactivatable GFP, to identify the mechanisms of localization and modes of transport. We will then take what we have learned from these studies and construct a quantitative model to test if the mobility parameters are sufficient to explain the patterns of protein localization and light-driven transport. Experiments in aim 3 are designed to identify the signals that tell the proteins to move using newly developed, expressible signal transduction sensors. Understanding the mechanisms of light-induced protein transport could reveal experimental strategies for testing adaptive or protective roles of transport directly and may lead to strategies for therapeutic intervention to slow or reverse photoreceptor degeneration in congenital disease. The proposed work fits into the National Plan for Eye and Vision Research under the Retinal Disease Program.